Photosynthetic and transcriptome responses to fluctuating light in Arabidopsis thylakoid ion transport triple mutant

Abstract Fluctuating light intensity challenges fluent photosynthetic electron transport in plants, inducing photoprotection while diminishing carbon assimilation and growth, and also influencing photosynthetic signaling for regulation of gene expression. Here, we employed in vivo chlorophyll‐a fluorescence and P700 difference absorption measurements to demonstrate the enhancement of photoprotective energy dissipation of both photosystems in wild‐type Arabidopsis thaliana after 6 h exposure to fluctuating light as compared with constant light conditions. This acclimation response to fluctuating light was hampered in a triple mutant lacking the thylakoid ion transport proteins KEA3, VCCN1, and CLCe, leading to photoinhibition of photosystem I. Transcriptome analysis revealed upregulation of genes involved in biotic stress and defense responses in both genotypes after exposure to fluctuating as compared with constant light, yet these responses were demonstrated to be largely upregulated in triple mutant already under constant light conditions compared with wild type. The current study illustrates the rapid acclimation of plants to fluctuating light, including photosynthetic, transcriptomic, and metabolic adjustments, and highlights the connection among thylakoid ion transport, photosynthetic energy balance, and cell signaling.


| INTRODUCTION
Plants use sunlight in the light reactions of photosynthesis to drive electron transport reactions in the thylakoid membrane of chloroplasts.The linear electron transport from photosystem II (PSII; P680) via cytochrome b 6 f complex (Cytbf) toward photosystem I (PSI; P700) ultimately produces reducing power (NADPH) in the chloroplast stroma and is coupled to H + translocation into the thylakoid lumen.
This leads to the build-up of a proton motive force (PMF) across the thylakoid membrane, composed of a pH (ΔpH) and an electric gradient (ΔΨ), driving ATP production at the ATP synthase.Together, NADPH and ATP are used for CO 2 fixation and other metabolic processes within the chloroplast.To grow and thrive in natural environments where sunlight intensity quickly and extensively fluctuates, for example, due to intermittent cloud cover and sun flecks (Kaiser et al., 2018), plants have evolved different light acclimation responses (Gjindali et al., 2021;Long et al., 2022).These responses are highly dependent on the light environment during plant growth as well as the frequency, duration, and intensity of fluctuating light (FL) conditions (Alter et al., 2012;Niu et al., 2023;von Bismarck et al., 2023;Yin & Johnson, 2000).Short-term responses also include photoprotective mechanisms (Allahverdiyeva et al., 2015) that are both rapidly inducible to prevent over-excitation during high light and rapidly reversible to ensure sufficient photochemistry under low light.
The most flexible photoprotective mechanisms in angiosperms are triggered by enhanced acidification of the thylakoid lumen.Increase in ΔpH triggers the regulated non-photochemical quenching (NPQ), which consists of mechanisms to dissipate excess light energy as heat (Ruban, 2016).These mechanisms are enhanced by the lumenal protonation of the PSBS protein and by the low pH-dependent interconversion of violaxanthin (V) to zeaxanthin (Z) in the xanthophyll cycle, mediated by violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE).Furthermore, lumen acidification slows H + -coupled electron transport through the Cytbf known as photosynthetic control (Tikhonov, 2014).This, in turn, leads to accumulation of oxidized PSI (P700 + ), which in itself is capable of harmlessly dissipating excess energy as heat (Schlodder et al., 2005).
Lumen pH-dependent photoprotection is modulated by the influx/efflux of H + and other ions (K + , Mg 2+ , and Cl À ) through thylakoid ion channels and transporters.Ion exchange across thylakoid membranes directly modifies the ΔΨ component of the PMF and also adjusts the ΔpH, thereby integrating electron transport, NPQ, and ATP synthesis (Spetea et al., 2017).Rapid acidification of the lumen and subsequent induction of NPQ under high light requires influx of Cl À counter-ions through the voltage-dependent chloride channel VCCN1, which leads to a decreased ΔΨ and consequently an increased ΔpH (Duan et al., 2016;Herdean et al., 2016).A similar function through the efflux of K + co-ions has been reported for the two-pore potassium channel TPK3 (Carraretto et al., 2013), although its relevance for the regulation of photosynthesis is still debated (Höhner et al., 2019).The Cl À channel/transporter CLCe is suggested to regulate thylakoid Cl À homeostasis, ATP synthase activity, and electron transport during low light (Dukic et al., 2022;Herdean et al., 2016).The major pathway for H + efflux from the lumen is through the ATP synthase complex, which is known to impact the formation and relaxation of ΔpH-dependent NPQ (Kanazawa et al., 2017), whereas additional H + efflux through the K + /H + antiporter KEA3 accelerates NPQ downregulation after high light phases by decreasing ΔpH and increasing ΔΨ component of the PMF (Armbruster et al., 2014).VCCN1, CLCe, and KEA3 are known to function independently to adjust photosynthesis in response to changes in light intensities (Dukic et al., 2019;Li et al., 2021;von Bismarck et al., 2023).
Photoprotective mechanisms decrease the efficiency of electron transport and CO 2 assimilation (Long et al., 2022;Murchie & Ruban, 2020) and impact the generation of reactive oxygen species (ROS) and other signals in the chloroplast, which modulate the expression of chloroplastic and nuclear genes (Gollan & Aro, 2020).
In the current study, we explored the interactions between energy balance in photosynthetic light reactions, global gene expression, and metabolism in Arabidopsis thaliana wild type (WT) and the triple mutant lacking KEA3, VCCN1, and CLCe (Dukic et al., 2019) exposed to 6 h FL and constant light (CL) treatments.FL conditions greatly enhanced photoprotective energy dissipation of both photosystems, albeit with altered kinetics leading to moderate PSI photoinhibition in kvc.FL-induced gene expression changes in WT were associated particularly with secondary metabolism and biotic stress, whereas in kvc, the corresponding upregulation of biotic stress gene expression occurred already during growth under CL conditions, suggesting an indirect link between thylakoid ion channels/transporters and biotic stress response signaling.

Arabidopsis thaliana
Columbia-0 (WT) plants and the kea3-1vccn1-1clce-2 (kvc) triple loss-of-function mutant along with respective single mutants clce, vccn1, and kea3 (Dukic et al., 2019) were grown for 5 weeks in soil in short-day conditions (8 h light/16 h darkness) at 23 C and 50% humidity under 100 μmol photons m À2 s À1 and watered three times a week with tap water.FL treatments were carried out using an LED array (Heliospectra, Sweden) set to alternate between low light (LL, 50 μmol photons m À2 s À1 for 4 min) and high light (HL, 500 μmol photons m À2 s À1 for 1 min).Fiveweek-old plants were treated with FL for 6 h.Control plants were exposed to CL at 100 μmol photons m À2 s À1 under the same LED array for 6 h.Plants were shifted to CL or FL conditions at the beginning of the photoperiod so that sample collection was performed toward the end of the photoperiod.Sample collection for all experiments was performed at the same time of day to minimize effects on photosynthetic performance and gene expression (Schneider et al., 2019).Long-term FL effects on thylakoid protein abundances in WT and kvc were additionally investigated from plants grown under an FL regime for 6 weeks (same conditions as above).

| In vivo chlorophyll-a fluorescence and P700 difference absorption measurements
Simultaneous in vivo chlorophyll a fluorescence and P700 difference absorption measurements were performed from each genotype using a Dual-PAM 100 (Walz, Germany) on mature leaves of CL-and FLtreated plants.Measurements were performed with fluorescence measuring light intensity below <1.0-μmol photons m À2 s À1 and maximum P700 measuring light intensity.Red light (635 nm) was used for actinic illumination for all measurements, including saturating pulses (SP, 700 ms, 8000 μmol photons m À2 s À1 ).Far-red light (720 nm) intensity was set to 130 μmol photons m À2 s À1 .PSII quantum yields and parameters were calculated as follows (Hendrickson et al., 2004;Kramer et al., 2004): maximum quantum yield of PSII photochemistry, , with F 0 0 estimated according to Oxborough and Baker (1997).PSI quantum yields were calculated as follows (Klughammer & Schreiber, 2008) et al., 1989) and average F v /F m from separate measurements after 30-min dark acclimation of leaves (see above).This approach exploits oxidation of electron transport chain through the preferential excitation of PSI with far-red light, which decreases F to the F 0 level in the absence of PSII photoinhibition.The validity of this approach was confirmed by comparing the F 0 level after 30-min dark acclimation and after 10 s far-red illumination, which showed no significant difference in fluorescence level (T-test, p < 0.05, n = 4; Figure S1).A similar approach was used for normalizing fluorescence traces, whereas P700 traces needed to be corrected for signal drift assuming a constant steady-state P700 oxidation level (P) at the end of each LL phase.

| RNA isolation and transcriptome analysis
Mature leaves were harvested immediately after 6 h CL or FL treatment from four different plants of each genotype and snap frozen in liquid nitrogen.Total RNA was isolated using the Innuprep Plant RNA Kit (Analytik-Jena, Germany).RNA libraries were prepared and sequenced on an Illumina HiSeq 2000 platform at BGI Genomics (China) and deposited in NCBI sequence read archive (PRJNA735049).Reads from RNA-seq were aligned to the A. thaliana reference genome containing gene annotations described in TAIR10, using Stand NGS software v3.4 (Avadis, India).Gene expression was quantified and normalized to the median expression value of all genes using the DESeq package (R).Differential expression of genes with log2 fold change (log2 FC) ≥ 1 was determined by a two-way ANOVA test.P-values were adjusted for false discovery rate (FDR) using the Benjamini-Hochberg procedure.
Gene ontology (GO) enrichment analysis was performed using the Gene Ontology Online Resource (http://pantherdb.org/webservices/go/overrep.jsp) to identify biological processes GO terms that were statistically significantly enriched (FDR corrected P < .05) in gene lists of interest.Non-redundant GO term lists were obtained by manually collapsing overlapping GO terms into a single term.Subcellular loci were predicted using the subcellular localization database (SUBA4; https://suba.live/).
The mobile phase flow rate was 1.6 mL min À1 .Peaks were quantified using authentic standards.

| Leaf starch content assay
Starch was isolated from mature leaves of 5-week-old WT and kvc plants subjected to 6 h CL or FL treatment.One hundred milligrams of leaf tissue was weighed and extracted three times by bead-beating in 800 μL of methanol:chloroform:water (12:5:3, v/v).The insoluble pellet containing starch was quantified using a total starch assay kit (Megazyme, Ireland).

| Cuticle permeability assay
A toluidine blue O (TBO) leaf drop assay was performed as described by Cui et al. (2019).Two mature leaves were harvested from each of eight individual WT and kvc that had been treated with either 6-h CL or FL.Five-micoliter drops of 0.05% TBO in acetate buffer (pH 4) were pipetted onto the adaxial surface of each leaf and incubated on wet paper towel for 2 h.Leaves were rinsed thoroughly with distilled water and photographed with a Canon DSLR camera.
The extent of TBO infiltration was quantified using ImageJ (https://imagej.nih.gov/ij/) to count the total number of pixels within each TBO-stained area.as well as mechanosensitive and gated ion channels in the thylakoid and other membranes, were also not DE by FL treatment in either WT or kvc (Data S1).GO terms enriched in the FL-upregulated genes of both WT and kvc included wax/fatty acid and terpene biosynthesis, and responses to hypoxia, herbivory, and biotic stress (Table 1).
Around 1000 genes were downregulated by FL treatment, of which 654 genes were common for WT and kvc (Figure 3a,b).GO terms significantly enriched among downregulated genes included DNA replication and regulation of salicylic acid signaling (Table 1).Full lists of DE genes and GO terms can be accessed from Data S1 and S2.
Comparison of the WT and kvc triple mutant transcriptomes revealed under CL conditions only around 140 DE genes, of which 102 were upregulated in kvc.After FL exposure, over 70% of these genes were also upregulated in WT plants, 45% were upregulated in kvc in the FL versus CL comparison, and around 30% were unresponsive to or downregulated by FL (Figure 3c,d).Intriguingly, these data indicate that a majority of genes with higher expression in kvc than in WT under CL conditions are actually part of the FL regulon.GO term analysis showed significant enrichment in the kvc-upregulated genes for responses to biotic stimuli, including defensive responses to fungus and bacteria (Data S2), terms that were found upregulated by FL in both WT and kvc (Table 1).Comparative analysis showed almost complete overlap between kvc-upregulated and FL-upregulated GO terms of WT (Figure 3c and Data S2), further suggesting commonality between FL-and kvc-responsive processes.
In-depth transcriptome analysis revealed upregulation of secondary metabolic pathways in both WT and kvc genotypes after FL exposure.In particular, enzymes involved in the metabolism of tocopherol and carotenoids, and those involved in long-chain fatty acid, wax, and suberin synthesis were upregulated (Table 2).Notably, nine genes encoding berberine bridge enzyme (BBE)-like proteins were upregulated after FL treatment, corresponding to 75% of the BBE-like enzymes with statistical significance in the current experiment (Table 2), and 35% of the entire BBE-like enzyme family in Arabidopsis (Daniel et al., 2017).BBE-like enzymes catalyze oxidation of various secondary metabolites, and some are involved in pathogen response (Benedetti et al., 2018).Several factors involved in synthesis and degradation of the terpenoid hormones abscisic acid (ABA) and gibberellic acid (GA) were DE by FL in both WT and kvc.Signaling activity of the fatty acid derivative hormone jasmonic acid (JA) appeared to be targeted by upregulation of several factors involved in JA degradation and turnover (Table 2).
To investigate possible similarities of the FL regulon with other biotic and abiotic stresses, genes upregulated by FL in WT and kvc were compared with published transcriptomic data.This analysis showed only 11%-12% overlap with genes upregulated after 60-min HL exposure (Figure S8; Gene Expression Omnibus [GEO] accession GSE94075; Crisp et al., 2017), whereas even lower overlap was observed with genes responsive to drought stress (GEO accession GSE65046; Bechtold et al., 2016;GEO accession GSE24177;Harb et al., 2010; not shown).On the other hand, around 40% of genes upregulated by flagellin peptide flg22, artificially triggering plant biotic response (GEO accession GSE5615; Qutob et al., 2006), were upregulated by FL in WT and/or the kvc mutant (Figure S8).
Taken together, these analyses revealed a strong upregulation in the expression of secondary metabolism and biotic stress-responsive genes in both WT and kvc, demonstrating that FL induction of these genes is mostly independent of KEA3, VCCN1, and CLCe in Arabidopsis.This was further supported by largely similar transcriptome response to short-term FL treatment of kea3, vccn1, and clce single mutants compared with both kvc and WT in upregulated GO-terms (Table S1) and differentially expressed genes (Table S2).However, although transcriptional changes associated with biotic stress defense were induced by FL in WT, kvc, and single mutants (Table S1), these were specifically upregulated in kvc as well as vccn1 and clce already under non-stress CL conditions, suggesting a potentially additive influence of altered thylakoid Cl À ion-homeostasis on the biotic stress response.
T A B L E 1 Gene ontology (GO) terms significantly enriched in upregulated or downregulated genes of WT and kvc.  to CL, with strong decrease of V and increases in the levels of antheraxanthin (A) and Z in both WT and kvc (Figure 4a).This resulted in a threefold increase in the de-epoxidation state between CL and FL in both genotypes (Figure 4b) with kvc showing a significantly higher ratio compared with WT.This analysis also demonstrated a small but significantly higher Z content in kvc than in WT, whereas total xanthophyll levels (V + A + Z) did not change after FL treatment (Figure 4a).The abundance of protochlorophyllide (Pchlide), a chlorophyll precursor, was substantially diminished after 6 h FL treatment as compared with CL conditions (Figure 4c), although the chlorophyll content and a/b ratios were between the genotypes (Figure S9A,B).

| FL causes a decrease in starch contents of leaves
The observed effects of FL on photosynthesis prompted us to investigate starch accumulation after 6 h FL exposure in WT and kvc leaves.
Relative starch content per fresh weight after FL exposure was approximately 50% lower in WT and kvc, whereas there was no difference between the two genotypes under either condition (Figure 5).

| Cuticle analysis
The observed upregulation by FL treatment of genes involved in wax and fatty acid synthesis pathways (Table 2) prompted an investigation of the integrity of the leaf cuticle.A leaf drop assay using toluidine blue (TBO) demonstrated significantly higher cuticle permeability in the kvc mutant treated with either CL or FL in comparison with WT leaves (Figure 6).et al., 2004;Kramer et al., 2004), the latter also including the formation of chlorophyll triplet states ( 3 Chl) and singlet oxygen ( 1 O 2 ) leading to PSII photoinhibition.For PSI, the dissipation via pathways associated with photoinhibition is Y NA , which estimates the reduced P700 fraction or PSI acceptor-side limitation (Klughammer & Schreiber, 2008), leading to ROS production and causing oxidative damage at the iron-sulfur clusters within PSI (Tiwari et al., 2016).
The downregulation of the quantum yields associated with oxidative damage is driven by the progressive upregulation of photoprotective dissipation pathways in both photosystems, which are triggered concomitantly with an increase of lumen acidification (ΔpH) during the HL phase.The increase in ΔpH leads to activation of energydependent NPQ mechanisms (qE) enhanced by both PSBS and Z (Sacharz et al., 2017) causing the subsequent increase in Y NPQ .In parallel, the increase in ΔpH also leads to increased photosynthetic control, resulting in a slow-down of linear electron transfer at the Cytbf complex (Tikhonov, 2014).This caused donor-side limitation of PSI and accumulation of oxidized PSI (P700 + ), resulting in the subsequent increase in Y ND and harmless dissipation of excess energy as heat by P700 + (Schlodder et al., 2005).It is noteworthy that the regulation of energy distribution in both photosystems during the short HL phase (1 min) of the FL-regime did not lead to adjustments of respective effective quantum yields of photochemistry (Y II , Figure 2a; Y I , Figure 2d), suggesting that the immediate regulation response of energy distribution focuses directly on minimization of photodamage rather than adjusting linear electron transport, typically observed under longer FL regimes (Ikeuchi et al., 2014).In the kvc mutant, regulation of photoprotective energy dissipation of both photosystems was clearly impeded in comparison with WT.This is in line with previous results showing that kvc has an altered partitioning of the PMF into ΔpH and ΔΨ (Dukic et al., 2019;Li et al., 2021).In kvc, this led to lower Y NPQ (Figure 2c), due to lower induction of NPQ (Figure S4A), and lower Y ND during HL (Figure 2F) as well as slower NPQ relaxation during the HL-LL transition compared with WT after both CL and FL treatments (Figures 2c and S4A).These differential features in kvc are ascribed to the lack of VCCN1 consequent loss of Cl À import activity into the lumen, leading to lower ΔpH during the HL phase (Duan et al., 2016;Herdean et al., 2016), as well as to the lack of 4.2 | The ion channel/transporter mutant kvc is sensitized to PSI photoinhibition during short-term FL treatment Impaired regulation of the photoprotective energy dissipation of both photosystems in kvc led to reduction of maximal PSII and PSI activity after 6 h FL treatment, marked by a 2% decrease of F v /F m (Figure 1a) and a 27% decrease of ΔP m (Figure 1b) as compared with WT.This suggests that the decreased NPQ response of kvc was not enough to overwhelm the constantly active PSII repair cycle (Chow & Aro, 2005), therefore accumulating only negligible amount of PSII photoinhibition.Because PSI lacks an efficient repair cycle, the accumulation of PSI photoinhibition is expected and routinely observed under FL conditions also in WT (Kono et al., 2014;Suorsa et al., 2012;Wei et al., 2021).However, the extent of PSI photoinhibition strongly depends on the duration of the FL treatment as well as on the applied interval frequency and used high and low light intensities (Kono & Terashima, 2014;Sejima et al., 2014;Tan et al., 2021;Tikkanen & Grebe, 2018), complicating any direct comparisons on PSI photoinhibition and associated thylakoid protein reorganizations between experiments using different FL treatments.Nevertheless, the 6 h FL treatment employed in this study was comparably short and expected to not induce major changes at thylakoid protein level.For this reason, the WT and kvc were grown for 6 weeks under the same FL treatment, followed by an assessment of photosynthetic protein contents.
These showed lower PSI content in kvc (Figure S7), which further underscored the susceptibility of PSI in kvc to PSI photoinhibition in comparison with WT, a phenomenon not observed under constant growth light conditions (Dukic et al., 2022).
It should be noted that in addition to kvc, also clce, vccn1, and kea3 diverse functions of thylakoid ion channels/transporters regulating photosynthesis (Armbruster et al., 2017;Spetea et al., 2017), the interaction of VCCN1 with CLCE and KEA3 likely explains the slightly enhanced FL-induced photoinhibition of PSI in kvc compared with individual single mutants.Specifically, during the LL-HL transition, the absence of VCCN1 (Duan et al., 2016;Herdean et al., 2016) would lead to a lower ΔpH and diminished photosynthetic control, which in turn results in more electrons reaching already acceptor-side limited PSI, hence causing ROS production and PSI photoinhibition during the HL phase (Huang et al., 2019;Tikkanen & Grebe, 2018).It is important to note that PSI photoinhibition is not per-se detrimental to photosynthesis, because only a drastic decrease in the active PSI content would limit linear electron transport (Schöttler & T oth, 2014), because of inherent fast forward electron transport reactions within PSI (Brettel & Leibl, 2001).Furthermore, PSI inhibition is mainly caused by an overreduction of the PSI acceptor side, effectively reducing the maximal PSI activity (Sonoike, 2011), which in turn decreases PSI acceptor-side limitation by increasing the availability of acceptors per PSI.This selfregulated adjustment likely explains the observed decrease in Y NA (Figure 2e) and increase in Y ND (Figure 2f) in PSI photoinhibited kvc after FL treatment similar to WT at the end of the HL phase.Consequently, it can be argued that the moderate extent of PSI photoinhibition in kvc mutant did not prevent acclimation to short-term FL conditions, in line with previous observations (Dukic et al., 2019).
4.3 | Transcript abundance of photoprotection genes and changes in primary metabolism upon shortterm FL treatment In both WT and kvc, the NPQ response was strongly enhanced by 6 h FL treatment although to a different extent (Figure S4A).Simultaneously, transcript analysis of WT revealed moderate upregulation of the PSBS gene (NPQ4, log2 FC = 0.8-1.1)as well as NPQ1 and NPQ2 genes (NPQ1, FC = log2 0.7-0.9;NPQ2, FC = log2 0.6-0.et al., 2012;Wei et al., 2021).The higher accumulation of Z in kvc compared with WT is likely linked to the specific loss of KEA3 (von Bismarck et al., 2023).Accumulation of transcripts in both genotypes for β-hydroxylase (CHY1; Table 2), which converts β-carotene directly to Z (Rissler & Pogson, 2001), may have also contributed to observed increases in Z after the FL treatment (Figure 4a).Furthermore, enhanced transcript accumulation of several enzymes involved in terpene biosynthesis in both genotypes (Table 2) may support the production of xanthophylls and other terpenoids required for photoprotection during FL-induced light stress.The slightly higher accumulation of Z after FL treatment in kvc might have contributed to increase in NPQ during HL phase (Figures 2c and S4A) but might also be related to its high antioxidant capacity in the thylakoid membrane (Havaux, & Dall'Osto, L., and Bassi, R., 2007), counteracting increased ROS production caused by higher ΔΨ in the first seconds of the LL-HL transition (Johnson & Ruban, 2014), which normally activates VCCN1 and leads to re-partitioning of PMF toward ΔpH (Herdean et al., 2016).
Light stress, like other abiotic stress factors, is known to impact starch metabolism (Ribeiro et al., 2022) and could explain the diminished starch accumulation after 6 h FL treatment (Figure 5).Lower starch abundance also relates to enhanced stomatal closure during FL, which decreases CO 2 fixation and biomass accumulation (Matthews et al., 2018).In the current work, accumulation in 6 h FL-treated WT and kvc of gene transcripts for several enzymes involved in the metabolism of 2-phosphoglycolate (Table 2), the toxic metabolite produced during photorespiration (Busch, 2020), supports the concept that FL exposure stimulates photorespiration at the expense of CO 2 fixation due to stomatal closure.Decreased carbon assimilation during FL reduces the consumption of ATP, leading to an increase in the stromal ATP/ADP ratio and a decrease in thylakoid ATP synthase activity (Wei et al., 2021).This is in line with a clear decrease in abundance of ATP synthase subunit AtpF upon growth of Arabidopsis kvc mutant under FL conditions (Figure S7) and possibly also contributes to lumen acidification.Decrease in ATP synthase, together with increase in PSBS and VDE abundance (Figure S7), not observed in kvc under CL conditions (Li et al., 2021), might be part of the long-term FL acclimation responses of kvc.Lower ATP synthase contents have been generally associated with increase in PMF (Kanazawa et al., 2017;Rott et al., 2011), which in the case of kvc might compensate for the lack of PMF modulation via ion channels/transporters and in turn increase the ΔpH-dependent NPQ response after long-term FL acclimation.

| Signaling and stress responses upon shortterm FL treatment
The major response at transcript level to the 6 h FL treatment, in WT and kvc, comprised the upregulation of a number of genes involved in diverse secondary metabolism pathways (Tables 1-2), which were largely similar in clce, kea3, and vccn1 single mutants (Tables S1-S2).
This prompted us to focus the investigation on only WT and kvc.The simultaneous induction of herbivory and biotic stress response pathways in WT and kvc (Table 1), as well as specific components of JA signaling (Table 2), suggest the activity of oxylipin signaling under FL.Furthermore, the signaling effects of 12-oxophytodienoic acid (OPDA), a JA precursor, could likewise account for the upregulation of abiotic stress-related genes detailed in Table 2, being often ascribed to H 2 O 2 signaling (Gollan & Aro, 2020), but can also be activated by 1 O 2 (Ramel et al., 2012).Increased 1 O 2 production is also supported by upregulation of tocopherol synthesis genes during FL in both genotypes (Table 2).The main source of 1 O 2 in chloroplasts is over-excitation of PSII leading to 3 Chl formation (Khorobrykh et al., 2020), which is supported by accumulation of reduced (closed) PSII centers during the HL phase in both genotypes (Figure S4B).
Nevertheless, the apparently higher 1 O 2 levels during 6-h FL treatment, in comparison with CL, caused only a minor decrease in F v /F m (Figure 1a), implying that the rate of constitutive PSII repair kept pace with PSII photoinhibition.Albeit no change in total chlorophyll content (Figure S9A) was observed, lower Pchlide levels (Figure 4c) and lower expression of Pchlide reductase (POR) after FL treatment (Data S1) predict a reduced synthesis of 5-aminolevulinic acid, the rate-limiting step for chlorophyll synthesis (Wang et al., 2022), which might prevent accumulation of photoreactive tetrapyrrole metabolites, otherwise providing an opportunity for 1 O 2 production during the turnover of photosensitive chlorophyll (Wagner et al., 2004).Taking these results together, it is conceivable that, despite enhanced Surprisingly, only a limited set of genes was DE in the kvc mutant under CL as compared with WT (Data S1).Of these, the upregulated genes in kvc versus WT comparison showed a substantial correlation with genes upregulated by FL in both genotypes (Figure 3d).This finding was even more striking when analyzing the GO terms induced by the kvc mutation in CL, which described biotic stress, defense responses, and JA signaling (Data S2) and were found to overlap almost completely with GO terms induced by FL (Figure 3e).This implies that biotic stress-responsive signaling pathways induced by FL in WT were already partially active in kvc under CL (non-stress) conditions.Similar upregulation of GO-terms in clce and vccn1, but not kea3, already under CL conditions (Table S1) suggests that biotic stress-responsive signaling pathways might be linked to altered thylakoid Cl À ion-homeostasis, which should be further investigated.In kvc, this observation may also relate to the results of cuticle integrity analysis, which, despite upregulation of genes involved in synthesis of long-chain fatty acids (LCFAs), wax, and cutin in both genotypes (Table 2), did not reveal any changes in permeability after the FL treatment (Figure 6), yet a higher cuticle permeability was evident in kvc in comparison with WT after both CL and FL treatments.Therefore, the upregulated transcription of cuticle fortification genes under FL may relate to other FL-induced processes, such as decreased CO 2 fixation, altered stomatal conductance, or upregulation of biotic stress response.It is also possible that changes in cuticle permeability may appear at a later time point that was not investigated here.
The leaf cuticle is implicated in defense against biotic infection, both by presenting a physical barrier to microbes and through regulation of host defense signaling.Compromised cuticle integrity correlates with increased resistance against the fungal pathogen Botrytis cinerea, due to enhanced generation of ROS and primed sensitivity to pathogenesis (Cui et al., 2019;L'Haridon et al., 2011).Cuticle permeability detected in kvc in the current study, alongside upregulation of genetic pathways associated with biotic stress (Tables 1 and 2), suggests that pathogen resistance may be induced, to some degree, in kvc.In light of these observations, including high expression of JA regulators ILL5 and JOX2 in kvc under CL (Table 2), it is tempting to speculate that oxylipin signaling was active in kvc under CL conditions, although this calls for further investigation, also in single ion-channel/ transporter mutants.to unstable conditions found in nature.However, upregulation of FLinduced genes in kvc mutant under CL conditions suggests a so far unexplored indirect link between thylakoid ion channels/transporters and biotic stress response signaling that remains to be investigated.

| CONCLUSIONS
effective quantum yield of PSII photochemistry Y II = (F m 0 À F)/F m 0 ; quantum yield of non-regulated energy dissipation, Y NO = F/F m ; quantum yield of regulated NPQ, Y NPQ = (F/F m 0 ) À (F/ F m ); rate constant of NPQ, NPQ = (F m À F m 0 )/F m 0 ; and photochemical quenching parameter estimating the fraction of open PSII centers, photochemistry in PSI, Y I = (ΔP m 0 À ΔP)/ΔP m ; yield of PSI acceptorside limitation, Y NA = (ΔP m À ΔP m 0 )/ΔP m ; yield of PSI donor-side limitation, Y ND = ΔP/ΔP m .After CL or FL treatment, plants were dark acclimated for 30 min to determine minimum (F 0 ) and maximum (F m ) fluorescence directly with an SP, whereas ΔP m was determined with an SP after additional 10 s of far-red pre-illumination and used for normalization of fluorescence and P700 traces as well as calculation of PSII and PSI parameters during separate experiments without dark acclimation.In these experiments, plants were subjected to a simulated FL regime with SPs at different time points throughout the measurement.For these measurements, F 0 was estimated from fluorescence level during 10s far-red illumination of the ΔP m determination at the end of each experiment.This allowed estimation of F m from F 0 under far-red light by using a rearranged PSII quantum yield equation F m = F 0 /(1 À (F v /F m )) (Genty

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Figure1b).The FL treatment led to a small decrease (2%) in F v /F m in both WT and kvc as compared with CL treatment, which was slightly more pronounced in kvc.In contrast, FL treatment had no significant effect on ΔP m in WT, whereas it resulted in an average decrease of 27% in kvc as compared with CL conditions, suggesting a moderate amount of PSI photoinhibition in kvc.Similar results were also obtained for single mutants clce, kea3, and vccn1, which showed equal reduction of F v /F m (FigureS2A) and slightly less pronounced reduction of ΔP m (FigureS2B) compared with kvc, especially in clce.In the second experiment, WT and kvc plants were recorded during a single FL cycle by directly transferring plants to the Dual-PAM-100 without dark-acclimation to maintain the corresponding 6 h CL-and FLacclimation states of the plants (see Section 2).Fluo and P700 traces revealed overall slower responses in kvc mutant as compared with WT, and FL treatment further altered Fluo and P700 kinetics (FigureS3), suggesting rapid changes in energy distribution within PSII and PSI, especially during HL and after shift from HL to LL phase.These changes were further investigated by including saturating pulses in the single FL cycle, which allowed calculation of PSII and PSI quantum yields (Figure2).During the HL phase, a substantial decrease in effective quantum yield of PSII (Y II , Figure2a) was accompanied by an initial strong increase in quantum yield of non-regulated energy dissipation (Y NO , Figure2b), which was successively replaced by an increase in quantum yield of regulated NPQ (Y NPQ ) (Figure2c).Upon transition to the second LL phase, all PSII quantum yields subsequently returned to their previous levels.Although the PSII quantum yields generally followed a similar response, substantial differences between WT and kvc upon the light treatment were evident.During HL, kvc showed a slower rise in Y NPQ as compared with WT after CL treatment, whereas FL treatment systematically increased Y NPQ in both WT and kvc as compared with CL conditions (Figure2c).The lower Y NPQ in kvc compared with WT did not lead to changes in Y II during the HL phase, but rather to higher Y NO in both CL and FL

F
I G U R E 2 Changes in PSII and PSI quantum yields in WT and kvc after CL and FL treatments.Wild type (WT) and kvc triple mutant (kvc) plants were treated for 6 h with constant light (CL, 100 μmol photons m À2 s À1 ) or 6 h with fluctuating light (FL, 50-μmol photons m À2 s À1 for 4 min and 500 μmol photons m À2 s À1 for 1 min) immediately prior to measurements with a Dual-PAM 100 system and subjected to a single FL cycle without dark-acclimation (for details, see Section 2).(a) Effective quantum yield of photochemistry in PSII (Y II ); (b) quantum yield of nonregulated energy dissipation (Y NO ); (c) quantum yield of regulated non-photochemical quenching (Y NPQ ); (d) effective quantum yield of photochemistry in PSI (Y I ); (e) yield of PSI acceptor-side limitation (Y NA ); (f) yield of PSI donor-side limitation (Y ND ).Data represents mean with error bars indicating standard deviation (n = 3-4).

3. 3 |
Modulations in transcript accumulation of WT and kvc upon 6 h exposure to fluctuating light In order to explore the effects of short-term FL on gene expression, especially in the context of dynamic photosynthetic responses due to lack of ion channels/transporters, the transcriptomes of WT and the kvc mutant exposed for 6 h to either CL or FL were primarily analyzed.The expression of approximately 1500 genes was upregulated at least twofold (log2 FC ≥ 1) in the FL versus CL comparison in each genotype and 1145 of these genes were commonly upregulated in both genotypes (Figure 3a,b).Notably, KEA3, VCCN1, and CLCe were not differentially expressed (DE) above the established threshold when FL and CL treatments were compared in WT (Data S1).Similarly, the expression of other known K + , Cl À , and Mg 2+ channels/transporters, F I G U R E 3 Expression pattern and comparisons of genes upregulated in kvc in relation to wild type.(a) Hierarchically clustered heatmap showing gene expression levels, normalized to median level expression, in three replicates of wild type (WT) and kvc samples exposed to 6 h constant light (CL) or 6 h fluctuating light (FL).Shown are all genes with differential expression in WT or kvc (DE; FC ≥ log2 2, FC ≤ log2-2) after FL exposure, in comparison with CL exposure.Gene expression level is shown according to color scale.(b) Venn diagram analysis of overlap between genes upregulated or downregulated in the FL vs. CL comparisons in each genotype.Orange circles indicate corresponding gene clusters shown in the heatmap (a).(c) Hierarchically clustered heatmap showing expression of 102 genes found to be upregulated in kvc under CL conditions, compared to WT under CL.Color range indicates normalized gene expression level.(d) Venn diagram illustrating the overlap between 102 genes upregulated in kvc vs. WT and those upregulated in WT FL vs. CL and kvc FL vs. CL (shown in b).Orange circles indicate corresponding gene clusters shown in the heatmap (c).(e) Venn diagram illustrating the overlap between 16 non-redundant gene ontology (GO) terms found enriched in genes upregulated in kvc vs. WT and those found in WT FL vs. CL and kvc FL vs. CL.Full lists of genes and GO terms are included in Data S1 and S2.

3. 4 |
FL exposure impacts the xanthophyll cycle pigment and abundance of a chlorophyll precursor To further investigate the mechanism behind the observed effects of 6 h FL treatment on NPQ, the carotenoids and chlorophylls in mature WT and kvc mutant leaves were measured.The overall profile of xanthophyll pigments was clearly changed by FL exposure as compared Short-term FL treatment enhances photoprotective dissipation of excitation energy in both photosystems This study focused on the dynamic photosynthetic responses to fluctuations in photon flux density.The comparison of WT and kvc exposed for 6 h to either CL or FL treatment revealed common rapid and reversible changes in the energy distribution of PSII and PSI during a single simulated FL regime.During the LL-HL transition, these comprised in both WT and kvc of a rapid decrease in Y II (Figure 2a) and Y I (Figure 2d) and concomitant increase in Y NO (Figure 2b) and Y NA (Figure 2e), both of which were subsequently replaced by increasing Y NPQ (Figure 2c) and Y ND (Figure 2f) for the remainder of the HL F I G U R E 4 Abundance of xanthophylls and protochlorophyllide.(a) Content of violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) xanthophylls in WT and kvc leaves treated with 6 h constant light (CL) or fluctuating light (FL).(b) Abundance of quenching xanthophylls (Z + A) relative to total xanthophyll content (Z + A + V) in WT and kvc leaves treated with 6 h CL or 6 h FL.(c) Abundance of protochlorophyllide (Pchlide) in WT and kvc leaves treated with 6 h CL or 6 h FL.Data represent mean with letters indicating statistically significant groups (ANOVA, Tukey-HSD, P < 0.05, error bars denote SD, n = 5).F I G U R E 5 Starch accumulation in leaves after CL and FL treatments.Abundance of starch in WT and kvc leaves after 6 h exposure to either constant light (CL) or fluctuating light (FL) conditions.Data represent mean with letters indicating statistically significant groups (ANOVA, Tukey-HSD, P < 0.05, error bars denote SD, n = 3).phase.Although subsequent replacements were already present in CL-treated plants, they became more pronounced after the FL treatment in both WT and kvc.These results, together with similar responses of the single mutants kea3, vccn1, and clce (Figure S5-S6), underlined that plants actively enhance the photoprotective energy dissipation of both photosystems upon the FL treatment and in turn decrease the energy dissipation via pathways associated with photoinhibition.Y NO comprises the rate constants of fluorescence and unregulated basal dissipation processes within PSII (Hendrickson KEA3 and concomitant loss of K + /H + antiport activity leading to increased ΔpH during the HL-LL transition (Armbruster et al., 2014), which matched the corresponding responses observed in vccn1 and kea3 single mutants (Figures S5-S6).
single mutants showed mild PSI photoinhibition phenotypes after 6 h FL treatment (Figure S2B), suggesting that altered thylakoid ionhomeostasis might generally affect PSI susceptibility to photoinhibition, with a potential additive effect in the absence of different ion channels/transporters.Nevertheless, based on extensive research on the F I G U R E 6 Cuticle permeability in leaves after CL and FL treatments.Quantification of the pixels stained with toluidine blue (TBO) in leaves of WT and kvc treated with either 6 h constant light (CL) or 6 h fluctuating light (FL).Data represent mean with (*) indicating statistically significant differences (two-sample unequal variance t-test, P < 0.05, error bars denote SD, n = 16).
7), expressing the VDE and ZE enzymes responsible for de-epoxidation and epoxidation of xanthophylls, respectively, and similar transcript abundances were recorded in kvc after the FL treatment (Data S1).It is unlikely that changes in protein contents would be directly responsible for rapid changes in the NPQ response upon the short FL treatment.More likely, rapid NPQ changes might be caused by a transient decrease in lumen pH during the HL phase of the FL treatment, also explaining the observed higher accumulation of de-epoxidized xanthophylls after FL treatment in both WT and kvc (Figure 4a,b) (Alter NPQ induction by FL, PSII over-excitation and subsequent 1 O 2 signaling are likely major contributors to the FL regulation in both WT and kvc.Additionally, the mild PSI photoinhibition in kvc did not result in a specific transcriptome response compared to WT.Previously reported upregulation of chloroplastic iron chaperones FER1, FER3, and FER4 in PSI photoinhibited pgr5 mutant(Gollan et al., 2017) were found to be significantly upregulated (log2 FC = 1.2-1.7) in both WT and kvc after FL treatment (Data S1).This might be explained by relatively early photoinhibition of PSI during the FL treatment leading to rapid self-adjustment of energy distribution within PSI (see above) similar to WT (Figure2d-f), which terminated PSI-dependent ROS signaling and a transcriptomic response to PSI photoinhibition(Lima-Melo et al., 2021).
photosystems linked to thylakoid ion transport and lumen acidification.These findings are in line with enhancement of PMF partitioning to ΔpH by FL(Wei et al., 2021), yet the current study clearly demonstrates that these effects are activated already within a few hours of FL exposure.Acclimation responses in FL-exposed plants are likely involved in protecting PSII and PSI from photodamage induced by continuously alternating HL and LL phases.Because of the clear role of thylakoid H + gradient in FL response, we also investigated the acclimation of the kvc mutant (Dukic et al., 2019), which lacks both Cl À influx during LL-HL transition (VCCN1) and H + efflux during HL-LL transition (KEA3).In addition to the previously described effects on NPQ induction and relaxation, FL exposure of kvc induced a moderate extent of PSI photoinhibition, which is suggested to lead to selfregulated adjustments of PSI excitation energy distribution.FL treatment upregulated biotic stress response and secondary metabolism, including cuticle and terpenoid biosynthesis, which appear to involve 1 O 2 signaling pathway traced to PSII over-reduction that occurs during FL.Notably, neither PSII photoinhibition, chlorophyll turnover nor gene expression was substantially different between WT and kvc plants exposed to FL, which reiterates the robustness of acclimation